G-四联体与聚合酶链式反应联用可视化检测沙门氏菌  

Visual Detection of Salmonella by G-Quadruplex-Generating Polymerase Chain Reaction

作　　者：刘健慧 耿凤珍[2] 张先舟[1] 高浩 李聪[1] 高洁 檀建新[1] LIU Jianhui;GENG Fengzhen;ZHANG Xianzhou;GAO Hao;LI Cong;GAO Jie;TAN Jianxin(College of Food Science and Technology,Hebei Agricultural University,Baoding 071000,China;Affiliated Hospital of Hebei University,Baoding 071000,China)

机构地区：[1]河北农业大学食品科技学院,河北保定071000 [2]河北大学附属医院,河北保定071000

出　　处：《食品科学》2021年第22期331-338,共8页Food Science

基　　金：河北省自然基金重大项目(C2019204342);河北省科技支撑重大项目(16275505D);河北农业大学食品加工学科群项目(2020-06)。

摘　　要：建立对沙门氏菌的G-四联体与聚合酶链式反应(polymerase chain reaction,PCR)联用可视化检测方法。以沙门氏菌属特异性基因invH为检测目标,设计5’端含有G-四联体互补序列的上下游引物,通过PCR的特异性识别并扩增目标序列,获得大量含G-四联体的双链DNA,变性后与氯高铁血红素结合生成具有过氧化物酶活性的模拟酶(DNAzyme),并催化H_(2)O_(2)与2,2’-联氮-双(3-乙基-苯并噻唑琳-6-磺酸)二胺盐由无色变为绿色,实现G-四联体与PCR联用对沙门氏菌的可视化检测。在优化的检测体系下,沙门氏菌基因组的质量浓度对数与421 nm处的吸光度具有良好的线性关系,其回归方程为y=0.1299x+0.2179,R^(2)=0.9945,线性范围0.07~771.6 ng/μL,灵敏度为0.07 ng/μL。特异性分析表明:此方法适用于沙门氏菌属的检测,可成功应用于人工污染沙门氏菌牛奶的检测,检出限为1.2×10^(2) CFU/mL。通过检测30种市售样品,发现其结果与国标检测方法结果一致。本研究为食源性致病菌的检测提供了新的策略。In order to establish a visual detection method for Salmonella using G-guadruplex-generating polymerase chain reaction(PCR),the upstream and downstream primers containing G-quadruplex complementary sequences at the 5’end were designed according to the Salmonella-specific gene invH,which served as the target gene.Double-stranded DNA contained a large number of G-quadruplexes was obtained after specific amplification of the target sequences by PCR,and bound to hemin after being denaturated to generate DNAzyme with peroxidase-like activity.DNAzyme was able to catalyze the reaction between H_(2)O_(2) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt,resulting in color change from colorless to green and thus allowing visual detection of Salmonella.Under the optimized detection conditions,there was a good linear relationship between the logarithm of Salmonella genome concentration(y)and absorbance at 421 nm(x),which was fitted to the equation:y=0.1299x+0.2179(R^(2)=0.9945)with a linear range of 0.07–771.6 ng/μL,and the sensitivity was 0.07 ng/μL.The specificity analysis showed that this method was suitable for the detection of Salmonella,and could be successfully applied to artificially contaminated milk samples,with a detection limit of 1.2×10^(2) CFU/mL.By using this method,30 commercial samples were tested and the results were consistent with those obtained by the national standard detection method.This study provides a new strategy for the detection of foodborne pathogens.

关 键 词：G-四联体 聚合酶链式反应扩增 沙门氏菌 可视化检测 

分 类 号：TS201.6[轻工技术与工程—食品科学]